JP7244029B2 - Use of biosurfactants - Google Patents

Description

Techniques for ameliorating or treating mastitis and promoting milk production are disclosed.

Among dairy cow diseases, mastitis is a disease that occurs in the mammary glands and is directly related to milk productivity, and therefore has a very large economic impact. As a treatment for mastitis, administration of antibiotics is generally performed. However, when antibiotics are used, there are problems such as residue in the body, and the shipment of raw milk is restricted for a certain period of time, resulting in a large economic loss. Therefore, it is desirable to avoid the use of antibiotics as much as possible.

Against this background, there have been reports of attempts to prevent or treat mastitis by ingesting ingredients other than antibiotics (for example, ingredients derived from natural products) to dairy cows. For example, Patent Document 1 proposes a preventive/therapeutic composition for mastitis containing, as an active ingredient, a cultured composition obtained from a culture of a microorganism belonging to the genus Aureobasidium. In addition, Patent Document 2 proposes a preventive and therapeutic composition for livestock mastitis containing live baker's yeast as an active ingredient. It is envisioned that these compositions will be added to feed and administered to livestock, for example.

On the other hand, mammals, regardless of their species, are required to secrete or produce milk according to circumstances. In particular, milk of ruminant animals is widely used as foods either as it is or after being processed, and therefore there is a demand for improvement in its productivity. Conventionally, milk production has been improved mainly by crossbreeding using species with high milk production, improving growing environment, and improving feed.

On the other hand, biosurfactant is a general term for substances produced by microorganisms and having surfactant and emulsifying effects, and is reported to have high biodegradability and various physiological effects (Patent Document 3).

JP 2012-126657 A Japanese Unexamined Patent Application Publication No. 2001-224317 JP 2016-94398 A

One challenge is to provide new means for ameliorating or treating mastitis. It is also a challenge to provide new means for improving milk production.

As a result of intensive research to solve such problems, (1) biosurfactants have an ameliorating or therapeutic effect on mastitis, and (2) biosurfactants have an effect of promoting milk production or secretion. I found Based on such findings, further studies have been conducted, and the following inventions are provided.

Item 1
A mastitis ameliorating or therapeutic agent containing a biosurfactant.
Item 2
Item 1. The agent for ameliorating or treating mastitis according to Item 1, which is for ruminants.
Item 3
Item 3. The agent for improving or treating mastitis according to Item 1 or 2, which is locally applied to the breast.
Item 4
Item 4. The agent for ameliorating or treating mastitis according to Item 3, wherein topical application is continued for 3 days or more at a frequency of 1 time or more a day.
Item 5
A method of ameliorating or treating mastitis comprising topical application of a biosurfactant to the udder of a ruminant animal.
Item A
A milk production enhancer containing a biosurfactant.
Item B
The milk production enhancer according to item A, which is for ruminants.
Term C
The milk production promoter according to item A or B, which is topically applied to the breast.
Item D
A milk production enhancer according to paragraph C, characterized in that topical application is continued for 3 days or more at a frequency of 1 or more times a day.
Item E
A method of promoting milk production comprising topical application of a biosurfactant to the udder of a ruminant animal.
Term F
A method of promoting milk production according to Section E, wherein the frequency of topical application is once a day or more.

A means of ameliorating or treating mastitis is provided. Means are provided for promoting milk production or secretion. Thus, more efficient production of milk is possible.

Figure 2 shows the change in teat score of dairy cows with mastitis due to application of biosurfactant. Fig. 3 shows changes in teat firmness of dairy cows with mastitis due to application of biosurfactants. Changes in the number of somatic cells in the affected breast after application of a biosurfactant Fig. 3 shows changes in milk production of dairy cows due to application of biosurfactant.

The type of biosurfactant is not particularly limited and is arbitrary. Biosurfactants include, for example, mannosylerythritol lipids (MEL), mannosylmannitol lipids (MML), mannosylsorbitol lipids (MSL), mannosylarabitol lipids (MAraL), mannosylribitol lipids (MRL), cellobiose lipids, rhamnolipids, trehalose Lipids, sophorolipids, surfactins and the like are included. MEL, MML, MSL, MAraL, and MRL are also collectively referred to as mannosylalditol lipids (MAL). In one embodiment, the biosurfactant preferably has the ability to form lamellar structures and/or vesicles, eg MEL is preferred.

MEL has a structure represented by the following general formula (1).

(In the formula, R ₁ and R ₂ each independently represent an aliphatic acyl group having 2 to 24 carbon atoms, and R ₃ and R ₄ each independently represent a hydrogen atom or an acetyl group.)

MEL is classified into four types (MEL-A, MEL-B, MEL-C, and MEL-D) according to the types of R ₃ and R ₄ in general formula (1). In MEL-A, both R ₃ and R ₄ are acetyl groups. In MEL-B, _R3 is a hydrogen atom and _R4 is an acetyl group. In MEL-C, _R3 is an acetyl group and _R4 is a hydrogen atom. In MEL-D, both R ₃ and R ₄ are hydrogen atoms. A preferred MEL in one embodiment is preferably MEL-B.

In one embodiment, R ₁ and R ₂ in formula (1) preferably have 4 to 24 carbon atoms in the aliphatic acyl group, more preferably 8 to 14 carbon atoms, regardless of the type of MEL.

MEL has two optical isomers (4-O-β-D-mannopyranosyl-meso-erythritol (abbreviated as “4-O-β-MEL”) and 1-O-β with respect to the structure of the erythritol moiety. -D-mannopyranosyl-meso-erythritol (abbreviated as “1-O-β-MEL”)). In one embodiment the MEL is preferably 1-O-β-MEL and in another embodiment the MEL is preferably 4-O-β-MEL. In one preferred embodiment, MEL preferably has the structure of formula (2) below.

In MEL of formula (2), R ₁ and R ₂ are preferably aliphatic acyl groups having 4 to 24 carbon atoms, more preferably aliphatic acyl groups having 8 to 14 carbon atoms. In one embodiment, formula (1) MEL preferably has R ₃ is a hydrogen atom. In one embodiment, the MEL of formula (1) is preferred where _R4 is an acetyl group.

Biosurfactants are commercially available and can be obtained by chemical synthesis or by culturing microorganisms. In one embodiment, it is preferably produced by culturing microorganisms.

Examples of MEL-producing microorganisms include microorganisms belonging to the genera Pseudozyma, Moidiomyces, Ustilago, Sporithorium ni, Melanopsichium, and Kurthmanomyces. Preferred Pseudozyma microorganisms include Pseudozyma antarctica, Pseudozyma paratarctica, Pseudozyma rugulosa, Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma aphidis, Pseudozyma hubeiensis, and Pseudozyma tsukubaensis. Preferred Moidiomyces microorganisms include Moesziomyces antarcticus, Moesziomyces aphidis, and the like. Preferred Ustilago microorganisms include Ustilago hordei, Ustilago maydis, and the like. Preferred Sporisorium microorganisms include Sporisorium reilianum and Sporisorium scitamineum. Preferred Melanopsichium microorganisms include Melanopsichium pennsylvanicum. Preferred Kurthmanomyces microorganisms include Kurtzmanomyces sp. I-11 and the like. In a preferred embodiment, the MEL-producing microorganism is a Pseudozyma genus microorganism, more preferably a microorganism belonging to Pseudozyma tsukubaensis, more specifically Pseudozyma tsukubaensis 1E5 (JCM16987 strain), NBRC1940 (ATCC24555, CBS422.96 , CBS6389, DBVPG6988, PYCC4855, JCM10324, MUCL29894, NCYC1510, NRRLY-7792). These microorganisms may be used alone or in combination of two or more. Microorganisms belonging to Pseudozyma tsukubaensis selectively produce 1-O-β-MEL-B.

Production of MEL using microorganisms can be performed by any method and under any conditions. For example, MEL can be produced by culturing microorganisms. The medium to be used is not particularly limited, but it is desirable, for example, to use carbohydrates such as glucose, sucrose, blackstrap molasses, etc. as the carbon source. Fats and oils can also be used as carbon sources in addition to or in place of carbohydrates. The type of fats and oils is not particularly limited, and for example, vegetable oils, fatty acids or esters thereof can be added.

In one embodiment, it is preferred to add vegetable oil to the medium. The type of vegetable oil is not particularly limited, and can be appropriately selected according to the type of MEL intended. Vegetable oils and fats include, for example, soybean oil, olive oil, rapeseed oil, safflower oil, sesame oil, palm oil, sunflower oil, coconut oil, cocoa butter, and castor oil. Fatty acids include, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, behenic acid, nervonic acid, and the like. These fatty acids may be used alone or in combination of two or more. In one embodiment, a preferred fatty acid is oleic acid.

In one embodiment, MEL-producing microorganisms can be cultured in media containing only glucose as a carbon source. As the nitrogen source, an organic nitrogen source and an inorganic nitrogen source can be used in combination. For example, as an organic nitrogen source, one selected from the group consisting of yeast extract, malt extract, peptone, polypeptone, corn steep liquor, casamino acids, and urea, or two or more selected in combination can be used. As the inorganic nitrogen source, one or a combination of two or more selected from the group consisting of sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, and ammonia can be used. In another embodiment, a method for producing mannosylerythritol lipids is provided, comprising culturing a microorganism capable of producing mannosylerythritol lipids in a medium supplemented with fatty acid and glycerin.

The amounts of fatty acids and fats and oils are not particularly limited.

Culture conditions for microorganisms are not particularly limited. For example, it can be cultured for 3 to 7 days under conditions of pH 4-8, preferably pH 5-6, temperature 20-35°C, preferably 22-28°C. MEL can be recovered from the culture medium according to a conventional method.

The agent for improving or treating mastitis or the agent for promoting milk production may contain any other ingredients as long as they do not inhibit the action for improving or treating mastitis or the action for promoting milk production. Other ingredients include, for example, water; alcohols such as ethanol, tar pigments, coloring pigments such as iron oxide; preservatives such as parabens and phenoxyethanol; olive squalane, rice squalane, shark squalane. squalane such as dimethylpolysiloxane, silicone oils such as cyclic silicone; paraffin, liquid paraffin, vaseline, olefin oligomer, squalane and other hydrocarbons; jojoba oil, olive oil, macadamia nut oil, castor oil, safflower oil, sunflower oil, avocado Oil, vegetable oils such as canola oil, Chinese ginseng oil, rice germ oil, rice bran oil; glyceryl triacetylhydroxystearate, glyceryl triacetylricinoleate, glyceryl triisopalmitate, glyceryl triisostearate, glyceryl triundecanoate, trihydroxystearin Glyceryl acid, glyceryl trioleate, caprylic-capric triglyceride, caprylic-capric-myristic stearic triglyceryl, caprylic-capric-lauric triglyceryl, caprylic-capric-linoleic triglyceride Glyceryl, Glyceryl tricaprate, Glyceryl tallow, Glyceryl trimyristate, Glyceryl tristearate, Glyceryl tripalmitate, Glyceryl tri-2-heptylundecanoate, Glyceryl tribehenate, Glyceryl trimyristate, Glyceryl tricoate, Trilauric acid Triglycerides such as glyceryl, trilanolin fatty acid glyceryl, glyceryl 2-ethylhexanoate, and synthetic glycerides such as glyceryl trilinoleate; waxes such as beeswax, Japanese wax, carnauba wax; octyldodecyl myristate, cetyl palmitate, isostearyl isostearate, myristine Ester oils such as isopropyl acid; Higher alcohols such as cetanol, behenyl alcohol, isostearyl alcohol, jojoba alcohol, oleyl alcohol, stearyl alcohol, and long-chain branched fatty alcohol; Cholesterol, phytosterol, branched fatty acid cholesterol ester, macadamia nut fatty acid phytosteryl ester sterols and derivatives thereof; processed oils such as hydrogenated oils; higher fatty acids such as stearic acid, myristic acid, isotype long-chain fatty acids, and anteisotype long-chain fatty acids; ethers such as dicapryl ether; anionic surfactants such as sodium cetyl sulfate and N-stearoyl-L-glutamate; polyhydric alcohol fatty acid esters (excluding polyoxyethylene hydrogenated castor oil), modified silicones, sucrose esters, etc. cationic surfactants such as tetraalkylammonium salts; amphoteric surfactants such as betaine, sulfobetaine, and sulfoamino acid types; natural surfactants such as lecithin, lysophosphatidylcholine, ceramide, and cerebroside pigments such as titanium oxide and zinc oxide; antioxidants such as dibutylhydroxytoluene; inorganic salts such as sodium chloride, magnesium chloride, sodium sulfate and potassium nitrate; sodium citrate, potassium acetate, sodium succinate, asparagine Organic acid salts such as sodium acid, sodium lactate, gamma-aminobutyric acid, and lipoic acid; salts such as ethanolamine hydrochloride, ammonium nitrate, and arginine hydrochloride; chelating agents such as edetic acid; Neutralizing agents; biopolymers such as hyaluronic acid and collagen; placental extracts; ultraviolet absorbers such as hydroxymethoxybenzophenone sulfonate; vitamin A and derivatives thereof such as retinol, retinol acetate and retinol palmitate; Vitamin E and its derivatives such as tocopherol, delta tocopherol, tocopherol nicotinate, and tocopherol acetate; oil-soluble vitamin C derivatives such as ascorbyl palmitate, ascorbyl stearate, and ascorbyl tetraisostearate; xanthan gum, beta-glucan, oats, white fungus, etc. Polysaccharides extracted from, carrageenan and alginic acid, seaweed extracts such as agar, carboxyvinyl polymer, pectin, water-soluble polymers such as alkyl-modified carboxyvinyl polymer; dipropylene glycol, 1,3 butylene glycol, glycerin, polyhydric alcohols such as propylene glycol, sorbitol, malbitol, diglycerin, raffinose and hexylene glycol;

The agent for improving or treating mastitis or the agent for promoting milk production may be in any form as long as it can improve or treat mastitis or promote milk production. The agent for improving or treating mastitis or the agent for promoting milk production may contain only a biosurfactant, but in combination with any excipient that does not interfere with the action for improving or treating mastitis or the action for promoting milk production, It can be in any form suitable for parenteral administration, oral administration, or external administration (eg, pharmaceuticals, quasi-drugs, foods, cosmetics, veterinary drugs, veterinary foods, veterinary cosmetics). In one embodiment, the agent for improving or treating mastitis or the agent for promoting milk production is preferably in a form suitable for topical administration (e.g., external preparation), such as lotion, emulsion, gel, cream, ointment, It may be in a form selected from the group consisting of sprays, powders and the like. In one embodiment, the agent for ameliorating or treating mastitis or the agent for promoting milk production may be in a form suitable for non-topical administration, such as liquids, tablets, capsules, powders, granules, and the like. In another embodiment, the agent for improving or treating mastitis may be an injection.

The amount of the biosurfactant contained in the mastitis ameliorating or therapeutic agent or the milk production promoting agent is not particularly limited as long as the mastitis ameliorating or therapeutic action is exhibited, and is arbitrary. For example, an agent for improving or treating mastitis or an agent for promoting milk production may contain 0.1 to 30% by mass, preferably 0.5 to 20% by mass of biosurfactant.

Subjects to whom the agent for improving or treating mastitis or the agent for promoting milk production is applied are not particularly limited as long as the effect for improving or treating mastitis or the effect for promoting milk production is exhibited. For example, the subject is a mammalian animal. Preferably, the subject is a lactating female mammal. In one embodiment, the subject is a non-human mammal, preferably a ruminant. Ruminants include, for example, cattle, goats, sheep, deer, camels, bison, and the like. In view of their widespread livestock use, the subject is preferably selected from the group consisting of cattle, goats, sheep, horses and pigs, preferably cattle, more preferably dairy cows. In other embodiments, subjects may be pets such as dogs and cats.

In one embodiment, the mastitis ameliorating or therapeutic agent or the milk production promoting agent is preferably used by direct application to the breast and/or teat of the subject. Conditions such as application amount, application period, and application frequency are not particularly limited, and can be appropriately set according to the type, age, weight, time, and the like of the subject (for example, livestock). For dairy cows, for example, it is preferable to set the period (perinatal period, lactating period, etc.), stress state, nutritional state, age, and the like.

The mastitis ameliorating or therapeutic agent or the milk production promoting agent is preferably applied continuously over several days to several weeks. For example, 0.01 to 5 g, preferably 0.1 to 1 g per breast per day for 3 days or more, preferably 5 days or more, more preferably 8 days or more, still more preferably 10 days or more, continuously can be applied on a regular basis.

In one embodiment, the timing of applying the mastitis ameliorating or therapeutic agent or the milk production promoting agent can be set, for example, before or after wiping or milking.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.

Production example 1
Pseudozyma tsukubaensis (NBRC 1940) was vigorously cultured in YM medium at a culture temperature of 26° C. for 48 hours using a 500 ml Sakaguchi flask. The resulting solution was used as a seed culture medium, and cultured with aeration and stirring in YM medium (containing 5% olive oil) at a culture temperature of 26° C. for 7 days using a 10 L jar fermenter. An equal amount of ethyl acetate was added to the culture solution, and the mixture was distributed by stirring. An appropriate amount of anhydrous sodium sulfate was added to the ethyl acetate layer and allowed to stand still for 30 minutes, followed by concentration under reduced pressure and heating conditions to obtain crude MEL-B. This was subjected to silica gel column chromatography. The eluent was chloroform:acetone=1:0, chloroform:acetone=9:1, chloroform:acetone=1:1, chloroform:acetone=3:7, chloroform:acetone=0:1. The MEL-B fraction was fractionated and concentrated to obtain purified MEL-B. This was added to a 1,3 v-butylene glycol solution (50 v/v %) to a final concentration of 10 w/v % and dissolved to obtain a test solution.

Test example 1
After milking in the afternoon, 5 to 6 mL of the test solution was evenly applied to the udders and teats of 7 cows with udder inflammation. Milking was performed twice a day (morning and afternoon). This work was continued for 7 days. During and after this period, the nipple mouth score (method of Mein et al.) and the induration of the breast were measured using a muscle durometer (PPLS Digital Hardness Tester Type C, Pepareth Seisakusho Model No. TR-DHNC). As a result, as shown in FIG. 1, application of MEL improved the average nipple mouth score from 2.4 to 1.0 (healthy state). In addition, as shown in FIG. 2, the degree of udder hardening measured by a muscle hardness tester decreased from 1.13 to 0.06 (udder hardness of healthy dairy cows). Furthermore, when somatic cell counts were measured by fluorescence optical somatic cell assay, MEL application significantly reduced somatic cell counts after 21 days, as shown in FIG. Thus, it was confirmed that mastitis was significantly improved by biosurfactants represented by MEL.

Test example 2
After milking in the afternoon, 4 cows were uniformly coated with 5 to 6 mL of the test solution per udder on the udders and teats. Milking was performed twice a day (morning and afternoon). This operation was continued for 7 days, and the amount of milk per cow/day was measured. FIG. 4 shows the changes in the average measured milk yield. As shown in FIG. 4, it was confirmed that the application of MEL increased the amount of milk produced by dairy cows, and that the effect of MEL to increase the amount of milk was maintained even after the application of MEL was stopped. .

Claims (6)

A milk production enhancer containing mannosylerythritol lipids . The milk production promoter according to claim 1, which is for ruminant animals. 3. Milk production promoter according to claim 1 or 2, characterized in that it is topically applied to the breast. 4. The agent according to claim 3, characterized in that topical application is continued for 3 days or longer with a frequency of 1 or more times a day. A method of promoting milk production comprising topically applying a mannosylerythritol lipid to the udder of a ruminant animal. 6. A method of promoting milk production according to claim 5, wherein the frequency of topical application is once a day or more.

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